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Yes / α2,8-Linked polysialic acid (polySia) is an oncofoetal antigen with high abundance during embryonic development. It reappears in malignant tumours of neuroendocrine origin. Two polysialyltransferases (polySTs) ST8SiaII and IV are responsible for polySia biosynthesis. During development, both enzymes are essential to control polySia expression. However, in tumours ST8SiaII is the prevalent enzyme. Consequently, ST8SiaII is an attractive target for novel cancer therapeutics. A major challenge is the high structural and functional conservation of ST8SiaII and -IV. An assay system that enables differential testing of ST8SiaII and -IV would be of high value to search for specific inhibitors. Here we exploited the different modes of acceptor recognition and elongation for this purpose. With DMB-DP3 and DMB-DP12 (fluorescently labelled sialic acid oligomers with a degree of polymerisation of 3 and 12, respectively) we identified stark differences between the two enzymes. The new acceptors enabled the simple comparative testing of the polyST initial transfer rate for a series of CMP-activated and N-substituted sialic acid derivatives. Of these derivatives, the non-transferable CMP-Neu5Cyclo was found to be a new, competitive ST8SiaII inhibitor.

Sol-gel processing has been demonstrated to produce supported inorganic and hybrid microporous membranes with controlled physical and chemical properties under mild conditions. In preparing asymmetric membranes on mesoporous ceramic supports using traditional sol-gel processes, however, infiltration of the final coating material from smaller nanoparticles into the porous support can lead to unpredictable membrane thicknesses, poor reproducibility and reduced flux for separations.Herein we describe a size exclusion approach to prepare membranes by depositing well-defined relatively monodisperse particles on a mesoporous ceramic support. Ensuring that the particles remain on the surface by size exclusion can reduce or even eliminate infiltration. But if the porosity of the membrane top-layer is going to be finer than that of the support, it must be possible to sinter the particles to eliminate the interstitial porosity. Low temperature sintering is accomplished by preparing relatively compliant polysilsesquioxane particles through the introduction of organic substituents into the network of particles.To prepare membranes by size exclusion, we developed a sol-gel route to synthesize bridged polysilsesquioxane particles by polymerizing a dilute solution of monomers below their gelation concentration. Dynamic light scattering was used to monitor the particle size and size distributions during polymerizations up to the formation of gels. A membrane top-layer was successfully coated on a mesoporous titania-zirconia support through size exclusion of octylene- bridged polysilsesquioxane particles. To assist in determining if infiltration into the support has occurred and if particles are size-excluded from penetrating the support, we have covalently modified polysilsesquioxane particles with a fluorescent dye to provide direct visual evidence of the location of particles in the ceramic membrane. This is the first report of fluorescent diagnostics being used to detect infiltration and verify size exclusion of particles in asymmetric membrane deposition. We further created supported membranes of poly(phenylsilsesquioxane) through size exclusion of particles deposited on the support and then cured to establish a glassy, defect-free membrane coating without infiltration upon thermal exposure. Infiltration was verified with fluorescent dyes covalently bound into the particles. The size exclusion approach combined with fluorescent diagnostics allowed for the simplification of membrane formation and elimination of infiltration.

The most abundant biological thiols, homocysteine (Hcy), cysteine (Cys) and glutathione (GSH) have been the subject of intense research due to their association with a wide range of diseases. They play a key role in maintaining the redox status of biological systems. Selective detection methods for these thiols are challenging due to their similar structures and properties. Current commercially available detection methods use separations, fragile and expensive enzymatic or immunogenic materials and complex instrumentation. This has led to a global effort towards developing simple and inexpensive optical probes and indicators selective for specific biological thiols. Highly selective chemical probes and simple methods for detection and potential quantification of Hcy and GSH in their natural biological media have been developed. These indicators and methods are relatively simple and inexpensive for potential application at point of care. The selective detection of Hcy using novel asymmetric viologen chemical probes at room temperature is described as well as the use of commercially available materials under photochemical conditions. These probes respond linearly proportional to increasing Hcy concentrations, potentially enabling the monitoring of Hcy levels in human plasma. Additionally, new methods for the selective determination of GSH in human plasma, as well as its quantification in whole blood deposited on filter paper (dried blood spots), is also presented herein.

Les techniques de fluorescence sont des outils de choix pour l’étude et la compréhension fine des processus biologiques. Ceci requiert toutefois l’utilisation de sondes fluorescentes parfaitement adaptées au but visé et répondant aux différentes exigences requises pour l’application visée. Dans ce cadre, nous nous sommes plus particulièrement intéressés à l’élaboration de sondes biphotoniques de pH adaptées à une mesure très sensible de faibles variations de pH autour du pH neutre. Les variations et gradients de pH sont en effet impliqués dans un certain nombre de processus biologiques importants et peuvent être associées à des dysfonctionnements liés à certaines maladies. Dans ce cadre, nous avons développé de nouvelles sondes fluorescentes de pH fluorescentes présentant à la fois un comportement ratiométrique, une forte sensibilité autour du pH neutre et facilement excitables dans le proche IR par absorption à deux photons. Ces sondes de structure quadrupolaire et bolamamphiphile permettent ainsi la détection ratiométrique du pH dans des environnements biologiques au moyen d'une excitation biphotonique dans le proche IR. En parallèle, nous nous sommes intéressés à l’élaboration de nanoparticules hyperbrillantes dédiées à l’imagerie biologique par microscopie de fluorescence induite par excitation à deux photons. Nous nous sommes plus particulièrement attachées au design de nanoparticules organiques fluorescentes constituées de molécules organiques de bas poids moléculaire (FONs). Cette approche offre en effet une grande flexibilité et la possibilité d’accéder à des nanosondes ayant des brillances comparables aux très populaires quantum dots mais moins toxiques et plus facilement dégradables. L’ingénierie moléculaire des fluorophores utilisés pour la préparation des FON est cruciale puisqu’elle influence fortement à la fois les propriétés photophysiques (brillance, couleur…) et leur propriétés physico-chimiques (stabilité chimique et structurale, stabilité colloïdale). Dans ce contexte, une librairie de nouveaux chromophores dipolaires a été synthétisée et utilisées pour la préparation de FON par la méthode de nano-précipitation. Leurs propriétés ont été étudiées afin de déterminer la relation entre la structure du chromophore et les propriétés globales des nanoparticules constituées de ces colorants. Ce travail a permis d’identifier les paramètres structuraux permettant d’accéder à des nanoparticules présentant à la fois une brillance exceptionnelle, une émission modulable du vert au rouge et proche IR et une remarquable stabilité colloïdale. Ces nanoparticules présentent des potentialités majeures pour l’imagerie in vivo par excitation et détection dans le proche IR. / Fluorescence-based techniques are popular tools for the study and understanding of biological processes. This has prompted continuous research aimed at the development of a wide range of fluorescent probes specifically designed for specific applications. Among them, fluorescent pH probes are of much interest as pH variations or gradients are involved in many biological events and anomalous alterations are often related to the onset of dysfunctions and diseases. In this framework we have developed a series of promising two-photon pH fluorescent molecular probes. These quadrupolar bolaamphiphilic probes are of great interest, as they combine a steep pH dependence of their optical properties close to neutral pH, ratiometric behavior and large response to two-photon (2P) excitation in the NIR region. As such they offer much promise for ratiometric detection of the pH in biological environments and in situ monitoring of acidification. In parallel, we have been interest in the design of ultrabright nanoparticles for bioimaging purpose (in particular highly sensitive optical imaging). We chose to focus on Fluorescent Organic Nanoparticles made of organic molecules with low molecular weight (FONs) as they offer a flexible route and promising alternatives to toxic quantum dots. In this case the design of the dye used as building blocks of the FONs is of crucial importance and strongly influence the chemical and physical properties of the nanoparticles generated, such as their one and two-photon brightness and both their structural and colloidal stability. In that context a library of novel dipolar chromophores have been synthesized and used to prepare FONs using the nanoprecipitation method. Their properties were thoroughly investigated in order to determine the relationship between the molecular design of the isolated dye and the overall properties of the nanoparticles made of these dyes. As a result, Hyperbright FONs emitting in the green to NIR region and combining giant brightness and remarkable stability have been achieved. They offer major promise for bioimaging based on both excitation and detection in the NIR region.

A hemoglobina de Glossoscolex paulistus (HbGp) é caracterizada por uma massa molecular de 3.600 kDa, alta estabilidade oligomérica, resistência a auto-oxidação, e alta afinidade em ligar oxigênio. A estrutura quaternária desta proteína apresenta 144 cadeias com grupo heme (globinas) e 36 cadeias sem grupo heme (linkers), dispostos em duas camadas hexagonais. No presente trabalho, foi realizado o estudo da estabilidade da oxi-HbGp frente aos processos de dissociação oligomérica e desnaturação, utilizando duas classes de desnaturantes, ou seja, o surfactante brometo de dodeciltrimetilamônio (DTAB), e os agentes caotrópicos cloridrato de guanidina (GuHCl) e ureia. Convém mencionar ainda, que este estudo foi desenvolvido através do uso de duas sondas fluorescentes, 1-Anilino-8-naftaleno-sulfonato (1,8-ANS) e fluoresceína isotiocianato (FITC), usando as técnicas de absorção óptica, fluorescência estática, espalhamento de luz dinâmico (DLS) e fluorescência resolvida no tempo. Os resultados de fluorescência estática mostram que o DTAB induz um aumento na intensidade de emissão de fluorescência da sonda ANS, com o deslocamento do máximo de emissão para o azul de 517 para 493 nm. Duas transições são observadas, em 2,5 e 9,5 mmol/L de DTAB, e estão associadas à interação da sonda ANS com agregados pré-micelares e micelas, respectivamente. Na oxi-HbGp, ANS liga a sítios menos expostos ao solvente, quando comparado às micelas de DTAB, caracterizados pela emissão em 467-472 nm. A adição de DTAB ao sistema oxi-HbGp-ANS, no pH 7,0, induz a agregação da proteína, a dissociação oligomérica e desenovelamento da oxi-HbGp. No pH 5,0, a formação de agregados não foi observada. Além disso, o processo de desenovelamento induzido pelo DTAB apresenta duas transições, a primeira em virtude da dissociação oligomérica, e a segunda, provavelmente, devido à desnaturação das subunidades dissociadas. Por outro lado, GuHCl e ureia com concentrações acima de 1,5 e 4,0 mol/L respectivamente, induzem a desnaturação completa da oxi-HbGp, com redução dos grupos hidrofóbicos na superfície da proteína, e o deslocamento do ANS para o meio aquoso, detectado pela redução de intensidade de fluorescência. A técnica de fluorescência resolvida no tempo permitiu avaliar os valores dos tempos de vida para a sonda 1,8-ANS, bem como, para oxi-HbGp. Por último, a oxi-HbGp foi marcada com a sonda covalente fluoresceína isotiocianato (FITC) em dois valores de pH 7,0 e 9,0, e nas proporções sonda:heme de 1:5 e 2:1. A quantidade de FITC efetivamente ligada a oxi-HbGp por heme foi estimada a partir dos dados de absorção óptica. Supondo que o rendimento quântico de FITC no tampão é 100%, os rendimentos quânticos de FITC ligada a oxi-HbGp também foram encontrados. Além disso, estudos preliminares de dissociação e desnaturação da oxi-HbGp marcada com FITC, na presença do surfactante DTAB, foram realizados. / Glossoscolex paulistus hemoglobin (HbGp) is characterized by a molecular mass of 3,600 kDa, a high oligomeric stability, resistance to oxidation and a high affinity to oxygen. The quaternary structure of this macromolecule consists of 144 globin chains, and 36 additional chains lacking the heme group, named linkers, organized in a double-layered hexagonal structure. In this study, the oxy-HbGp stability, as well as, the oligomeric dissociation and unfolding processes were studied, using two types of denaturants,the surfactant Dodecyl-trimethylammonium bromide (DTAB), and chaotropic agents guanidine hydrochloride (GuHCl) and urea. Moreover, this study was developed based on 8-anilino-1-naphtalene-sulfonic acid (ANS) and fluorescein isothiocyanate (FITC) fluorescence probes, using the techniques of optical absorption, static fluorescence, dynamic light scattering (DLS) and time resolved fluorescence. The results of static fluorescence show that dodecyl-trimethylammonium bromide (DTAB) induces an increase in ANS fluorescence emission intensity, with maximum emission wavelength blue-shifted from 517 to 493 nm. Two transitions are noticed, at 2.50 and 9.50 mmol/L of DTAB, assigned to ANS interaction with pre-micellar aggregates and micelles, respectively. In oxy-HbGp, ANS binds to protein sites less exposed to solvent, as compared to DTAB micelles, characterized by emission at 467 - 472 nm. At pH 7.0, the addition of DTAB to the oxy-HbGp-ANS system induced the protein aggregation, oligomeric dissociation and unfolding of oxy-HbGp. At pH 5.0, no formation of aggregates was observed. Moreover, DTAB-induced unfolding process displays two transitions, one due to oligomeric dissociation and the second one, probably, due to the denaturation of dissociated subunits. On the other hand, guanidine hydrochloride (GuHCl) and urea, at concentrations above 1.5 and 4.0 mol/L, respectively, induce the full HbGp denaturation, with reduction of ANS-bound oxy-HbGp hydrophobic patches on the surface of the proteins. The shift of ANS to the aqueous medium was detected by the reduction in the fluorescence intensity. Time resolved fluorescence technique allowed to evaluate the lifetimes of ANS, as well as, for oxy-HbGp. Finally, oxy-HbGp was labeled with covalent probe fluorescein isothiocyanate (FITC), at pH values 7.0 and 9.0, and at probe: heme ratios of 1:5 and 2:1. The quantity of FITC effectively bound to oxy-HbGp, on the heme basis was estimated from the optical absorption data. Assuming a 100% quantum yield for FITC in buffer, the quantum yield of FITC bound to oxy-HpGp was also estimated. In addition, preliminary studies of dissociation and denaturation of oxy-HbGp labeled with FITC, in the presence of DTAB surfactant, were accomplished.

As espécies reativas de oxigênio (ROS) podem ser responsáveis por causar danos às membranas dos espermatozoides, fragmentação de DNA, entre outros fatores, influenciando assim na fertilidade principalmente no processo de criopreservação do sêmen. A melatonina (MEL) e o ácido ferúlico (AF) são potentes agentes antioxidantes que poderiam atuar no controle da produção de ROS no sêmen equino. Este estudo teve como objetivo avaliar o efeito dos antioxidantes AF e MEL na criopreservação do sêmen equino. Foram utilizados 5 ejaculados de 4 garanhões. Dentre os tratamentos aplicados, foram utilizadas duas concentrações de cada antioxidante (AF 0,5mM, AF 1,2mM, MEL 2mM e MEL 1µM) além do controle (diluidor de congelação convencional BotuCrio®), totalizando 5 tratamentos. As variáveis analisadas foram cinética espermática pelo sistema CASA (programa SCA - Sperm Class Analyser), morfologia, integridade de membranas plasmática, acrossomal e potencial de membrana mitocondrial, com o uso das sondas fluorescentes PI, Hoescht 33342, FITC-PSA e JC-1 além da produção de (ROS) pelo espermatozoide com a sonda fluorescente CellRox Deep Red®. Comparações entre os tratamentos foram realizadas pelo modelo linear generalizado (PROC GLM) do SAS (Versão 9.3) e as diferenças entre eles foram localizadas através do teste de Duncan. A probabilidade de P0,05 foi considerada como diferença significativa. Os resultados para características da motilidade tiveram diferença significativa em alguns aspectos, porém nenhum tratamento foi superior ao controle. Houve diminuição no percentual de defeitos maiores nas amostras tratadas com AF 1,2mM, MEL 2mM e MEL 1µM comparadas ao grupo controle. No que diz respeito à integridade de membranas, o tratamento MEL 1µM apresentou porcentagens significativamente melhores nas células com membrana plasmática intacta, acrossomo intacto e alto potencial de membrana mitocondrial, quando comparadas ao grupo controle. As células em estresse oxidativo não se diferenciaram entre os tratamentos. O uso da sonda fluorescente CellRox Deep Red® foi validado para espermatozoides de equinos. Foram utilizados 4 ejaculados de 4 garanhões aos quais eram submetidos aos tratamentos T0 (fração do ejaculado não submetida à indução do estresse oxidativo), T50 (50% da amostra não induzida e 50% induzida ao estresse oxidativo) e T100 (amostra induzida ao estresse oxidativo). Os dados de porcentagem de células positivas (com estresse oxidativo) foram submetidos à análise de regressão polinomial pelo modelo linear generalizado (PROC GLM) do SAS (Versão 9.3). O valor do coeficiente de determinação (R2) foi igual a 0,88 e a probabilidade de P0,05 foi considerada significativa. Pode-se concluir que o tratamento MEL 1µM contribui para a preservação da integridade de membranas espermáticas durante o processo de criopreservação do sêmen equino e que a sonda fluorescente CellRox Deep Red® é eficiente na detecção de espécies reativas de oxigênio no espermatozoide de garanhões. / Reactive oxygen species (ROS) can be responsible for causing damage to the membranes of sperm, DNA fragmentation, among other factors influencing fertility especially in cryopreservation. Melatonin (MEL) and ferulic acid (FA) are potent antioxidants that could act in the control of ROS production in equine semen. This study aimed to evaluate the effect of antioxidants AF and MEL in cryopreservation of equine semen. Five ejaculates from four stallions were used. Among the treatments, we used two concentrations of each antioxidant (AF 0.5mM, AF 1.2mM, MEL 2 mM and MEL 1µM) beyond the control (conventional freezing extender BotuCrio®), totaling five treatments. The parameters analyzed were sperm kinetics with the CASA system (SCA program - Sperm Class Analyzer), morphology, plasma and acrossomal membrane integrity mitochondrial membrane potential, using fluorescent probes PI, Hoechst 33342, FITC-PSA and JC- 1 over production ROS by the sperm with the fluorescent probe CellRox Deep Red®. Comparisons between treatments were performed by generalized linear model (PROC GLM) of SAS (version 9.3) and the differences between them were located with the Duncan test. The probability of P0.05 was considered significant. The results for the motility characteristics were significant differences in some aspects, but no treatment was superior to the control. There was a decrease in the percentage of major defects in the samples treated with AF 1.2mM, MEL 2 mM and MEL 1µM compared to the control group. Regarding to membrane integrity, treatment MEL 1µM showed significantly better in percentages of cells with intact plasma membrane, intact acrosome and high mitochondrial membrane potential compared to the control group. Cells with oxidative stress not differ between treatments. The fluorescent probe CellRox Deep Red® was validated for equine sperm previously. Was used ejaculates of 4 stallion which were subjected to the treatments T0 (fraction of the ejaculate not subjected to induction of oxidative stress), T50 (50% sample uninduced and 50% induced to oxidative stress) and T100 (sample induced to oxidative stress). The data of percentage of positive cells (with oxidative stress) were submitted to polynomial regression analysis based on generalized linear model (GLM PROC) of SAS (version 9.3). The value of the coefficient of determination (R2) was 0.88 and a probability of P0.05 was considered significant. It can be conclude that the treatment MEL 1µM contributes to the preservation of the integrity of sperm membranes during the equine sperm cryopreservation process and the fluorescent probe CellRox Deep Red® is efficient in the detection of reactive oxygen species in stallions sperm.

A degeneração testicular (DT) possui grande relevância dentre os distúrbios da reprodução e pode ser causada pelo aumento da temperatura testicular. Este provoca aumento do metabolismo celular, levando ao estresse oxidativo (EO) e apoptose. O tratamento usual consiste na retirada do agente causador e administração de antioxidantes; entretanto, pode não ser eficiente. Dessa forma, o presente estudo preconizou o tratamento da DT por meio de agentes com poder proliferativo: vitamina A e laserterapia de baixa intensidade (LTBI). Foram realizados três experimentos; o experimento 1 objetivou definir a dose de energia da LTBI necessária para a bioestimulação testicular. Foram utilizados seis carneiros distribuídos em três grupos: GC) insulação escrotal (IE) e sem tratamento (n=2); G28) IE e tratado com LTBI com 808 nm de comprimento de onda, 30 mW de potência e 28 J/cm² de densidade de energia por 15 dias a cada 48 horas (n=2); G56) IE e tratado com LTBI com 808 nm, 30 mW e 56 J/cm² por 15 dias a cada 48 horas (n=2). Foram feitas análises clínicas, reprodutivas e histopatológicas. Os dados foram submetidos à análise de variância (ANOVA) e teste de Tukey. Apesar da LTBI diminuir as taxas de espermatozoides com membrana acrossomal íntegra, esta foi eficiente em aumentar a população celular dos túbulos seminíferos no G28. Portanto, a LBTI provocou efeito bioestimulatório em testículos degenerados de carneiros. O experimento 2 objetivou validar a técnica de avaliação do EO espermático por meio da sonda fluorescente CellROX Deep Red®. Foram realizados dois experimentos; o primeiro utilizou ejaculados de três carneiros tratados em T0 (ejaculado não submetido à indução de EO), T50 (50% não induzido e 50% induzido ao EO) e T100 (submetido ao EO). Os dados foram submetidos à regressão linear. No segundo experimento foram utilizados 16 carneiros submetidos à IE. Foram feitas avaliações do EO antes e após a IE. Os dados foram submetidos à ANOVA e teste LSD de Fisher. O coeficiente de determinação foi de 0,728 e houve aumento do EO após a IE. Assim, a sonda CellROX® foi capaz de detectar o EO espermático. No experimento 3 foi proposto tratamento para a DT baseado na suplementação vitamínica ou LTBI. Foram utilizados 33 carneiros distribuídos em seis grupos: CC) sem IE e sem tratamento (n=5); CA) sem IE e tratado com vitamina A IM 120.000 UI/animal, duas vezes por semana durante três semanas (n=6); CL) sem IE e tratado com LTBI protocolo G28 (experimento 1) (n=5); IC) IE e sem tratamento (n = 5); IA) IE e tratado com vitamina A IM 120.000 UI/animal, duas vezes por semana durante três semanas (n=6); IL) IE e tratado com LTBI protocolo G28 (n=6). Foram realizadas análises clínicas, reprodutivas, hormonais e histopatológicas. Os dados foram analisados usando o procedimento de modelos mistos e os efeitos dos tratamentos foram avaliados utilizando contrastes ortogonais. Não houve efeito benéfico dos tratamentos para as características ultrassonográficas, qualidade espermática, concentração de testosterona e aspectos histopatológicos. Assim, os tratamentos não foram eficientes para melhorar a qualidade espermática nem promover a proliferação celular. / The testicular degeneration (TD) has great significance among the reproductive disorders and one of the main causes is the increase in testicular temperature. High testicular temperature results in increase cellular metabolism, leading to oxidative stress and apoptosis. The treatment consists in removing the causative agent and administration of antioxidants; however, it could be not efficient. The objective of this study is to recommend the treatment of TD by administering agents with proliferative action: vitamin A and low level laser therapy (LLLT). For this, three experiments were conducted. In experiment 1 the objective was to define the dose of energy for LLLT testicular biostimulation; it was used six rams distributed in three groups: GC) scrotal insulation (SI) and untreated (n=2); G28) SI and treated with LLLT with 808 nm, 30 mW and 28 J/cm ² of power density for 15 days every 48 hours (n=2); G56) SI and treated with LLLT with 808 nm, 30 mW and 56 J/cm² for 15 days every 48 hours (n=2). Clinics, reproductive and histopathological analyzes were done. Data were subjected to analysis of variance and Tukey test. The rates of sperm with intact acrosome membrane were decreased by LLLT, but the LLLT was effective in increasing the cell population of the seminiferous tubules in the G28. Thus, LLLT was able of causing stimulatory effect in degenerate testis of rams. The objective of experiment 2 was to assess the technique of evaluation of sperm oxidative stress (OS). This study was divided in two experiments; in experiment 1 was used ejaculates of three rams treated in T0 (ejaculate that was not submitted to OS induction), T50 (50% without OS and 50% inducted to OS) and T100 (entire submitted to OS induction). Data obtained were evaluated by linear regression analysis. In experiment 2, sixteen rams were submitted to SI. Analyses of OS were done before and after the SI. Data obtained were evaluated by analysis of variance and Fisher\'s LSD test. The determination coefficient was of 0.728 and there were increase in sperm showing OS after SI period. Thus, CellROX® fluorescent probe was able to detect sperm OS. The objective of experiment 3 was establish a treatment for TD based on vitamin A supplementation or LLLT; 33 rams were distributed in six groups: CC) no SI and non-treated (n=5); CA) no SI and treated with 120,000 IU/animal of IM vitamin A, twice a week for three weeks (n=6); CL) no SI and treated with LLLT G28 protocol (experiment 1) (n=5); IC) SI and untreated (n=5); IA) SI and treated with 120,000 IU/animal of IM vitamin A, twice a week for three weeks (n=6); IL) SI and treated with LLLT G28 protocol (n=6). Clinics, reproductive, hormonal and histopathological analyzes were performed. Data were analyzed using the mixed models procedure and treatments effects were evaluated using orthogonal contrasts. There was no beneficial effect of treatments for ultrasonographic characteristics, sperm quality, testosterone concentration and histopathological aspects. Thus, the treatments were not effective for improving sperm quality or promoting cell proliferation.

A criopreservação do sêmen resulta em danos à estrutura espermática, sendo nítida a importância da avaliação das partidas de sêmen antes de serem submetidas à inseminação artificial em tempo fixo (IATF). Todavia, nem sempre as avaliações convencionais do sêmen são suficientes para identificar partidas que possam resultar em baixa taxa de prenhez no campo, sendo necessária uma investigação mais profunda e acurada. Neste sentido, este experimento foi realizado com o objetivo de identificar partidas de sêmen que apresentam falhas na fertilidade, mesmo sendo aprovadas pelas avaliações convencionais. Foram realizadas análises convencionais (motilidade, vigor, concentração e morfologia espermática) de 72 partidas de sêmen de 22 touros antes da estação de monta. Destas, 55 partidas de 18 touros foram aprovadas para o uso na IATF, mas somente 28 partidas de 10 touros foram utilizadas. As partidas de sêmen utilizadas na IATF foram submetidas a outras avaliações: análise computadorizada da motilidade espermática (CASA), integridade das membranas plasmática e acrossomal e potencial de membrana mitocondrial (por sondas fluorescentes em microscopia de epifluorescência). Os dados foram analisados pelo Proc Mixed do SAS usando o Test T. As taxas de prenhez das diferentes partidas de sêmen variaram de 71 a 37%, sendo a média e desvio padrão das partidas de 55,57±7,57%. Os dados de fertilidade a campo permitiram a separação das partidas de sêmen como de Alta (>50% de prenhez) e Baixa (50% de prenhez) fertilidade, sendo comparadas quatro partidas de Alta e quatro de Baixa fertilidade. Os dados das características seminais de todas as partidas de sêmen foram submetidos à análise por boxplot e separados em quartis superior e inferior. Quando se comparou as partidas de Alta e Baixa fertilidade notou-se diferença na taxa de prenhez (p<0,01), mas não foi notada diferença para motilidade (p=0,91), vigor (p=0,63), concentração (p=0,27), número de espermatozoides por palheta (NEP, p=0,27), número de espermatozoides móveis e normais por palheta (p=0,18), defeitos maiores (p=0,17), defeitos totais (p=0,43), integridade de membrana plasmática (MPI, p=0,07), alto potencial de membrana mitocondrial (AP, p=0,94), motilidade total (MT, p=0,10), VCL (p=0,80), VSL (p=0,75), VAP (p=0,88), LIN (p=0,78), STR (p=0,71) e BCF (p=0,13). No entanto, foram encontradas diferenças entre os grupos (Alta e Baixa) para defeitos menores (p=0,05), espermatozoides com integridade das membranas plasmática e acrossomal e função mitocondrial (PIAIA)/Palheta (p=0,01), integridade de acrossomo (AI, p=0,03), motilidade progressiva (MP, p<0.01) e rápidos (p<0,01). Quando comparados os quartis superior e inferior das características seminais foram encontradas diferenças para concentração espermática (p<0,01), NEP (p<0,01), número de espermatozoides móveis por palheta (p<0,01), espermatozoides móveis e normais (p<0,01), defeitos maiores (p<0,01), defeitos menores (p=0,01), defeitos totais (p=0,05), MPI (p<0,01), AI (p<0,01), AP (p=0,03), PIAIA (p<0,01), PIAIA/palheta (p<0,01), mas esta divisão de grupos por quartis superior e inferior não apresentaram diferença sobre a fertilidade (p>0,05); sendo que somente. Entretanto, para MT (p<0,01) e MP (p<0,01) além da diferença entre os quartis foi notado efeito da fertilidade (MT, p=0,05 e MP, p=0,01), sendo maior para os de baixa fertilidade. Pode-se concluir que os padrões de qualidade de partidas de sêmen que apresentam fertilidade distinta podem ser semelhantes, além disso, que pode haver diferença na qualidade espermática entre as partidas que não influenciam a fertilidade. Desta forma, são necessárias outras análises mais acuradas para investigar as causas de falha na fertilidade de algumas partidas de sêmen. / Semen cryopreservation results in damage to sperm structure, and it is clear the importance of evaluating the semen batches before submitted it to timed artificial insemination (TAI). However, conventional semen evaluations are not always sufficient to identify batch that may result in reduced pregnancy rate in the field, requiring a more thorough and accurate investigation. Thus, this experiment was conducted in order to identify semen batches that have gaps in fertility, even being adopted by conventional assessments. Conventional analysis (motility, vigor, concentration and morphology) were performed of 72 semen batches from 22 bulls before the breeding season. Of these, 55 batches from 18 bulls were approved for use in TAI, but only 28 batches from 10 bulls were used. Semen batches used in TAI were subjected to further assessment: computer-assisted sperm analysis (CASA), integrity of plasma and acrosomal membranes and mitochondrial membrane potential (by fluorescent probes under epifluorescence microscopy). Data were analyzed by Proc Mixed of the SAS using Test \"T\". Pregnancy rates of different semen batches ranged 71-37%, and the mean and standard deviation of the batches was 55.57 ± 7.57%. Field fertility data allowed the separation of the semen batches as \"High\" (>50% pregnancy rate) and \"Low\" (50% pregnancy rate) fertility. It was compared four batches of \"High\" and four batches of \"Low\" fertility. Data of the seminal characteristics of all the semen batches were analyzed by boxplot and separated into upper and lower quartiles. When comparing the batches of \"High\" and \"Low\" fertility was noticed a difference in the pregnancy rate (p<0.01), but was not noticeable difference in motility (p=0.91), vigour (p=0.63), concentration (p=0.27), number of spermatozoa per straw (NEP, p=0.27), number of motile and normal sperm per straw (p=0.18), major defects (p=0.17), total defects (p=0.43) plasma membrane integrity (MPI, p=0.07), high mitochondrial membrane potential (AP, p=0.94), total motility (TM, p=0.10), VCL (p=0.80), VSL (p=0.75), VAP (p=0.88), LIN (p=0.78), STR (p=0.71) and BCF (p=0.13). However, differences were found between the groups (\"High\" and \"Low\") for minor defects (p=0.05), sperm with plasma and acrosomal membranes integrity and high mitochondrial membrane potential (PIAIA)/straw (p=0.01), acrosomal integrity (Al, p=0.03) progressive motility (PM, p <0.01) and rapid (p<0.01). When comparing the upper and lower quartiles of the seminal characteristics differences were found for sperm concentration (p <0.01), NEP (p <0.01), number of motile sperm per straw (p<0.01), motile and normal sperm (p<0.01), major defects (p<0.01), minor defects (p=0.01), total defects (p=0.05), MPI (p<0.01), AI (p<0.01), AP (p=0.03), PIAIA (p<0.01), PIAIA/straw (p<0.01), but this division groups by upper and lower quartiles showed no difference in fertility rate (p>0.05). However, to MT (p<0.01) and MP (p<0.01) the difference between quartiles of fertility effect was noted (MT, p=0.05 and MP, p=0.01), been higher for low fertility. It can be concluded that patterns of semen quality of batches that have distinct fertility may be similar, furthermore, there may be differences in sperm quality among batches that do not affect fertility. Thus, it takes other more accurate analysis to investigate the causes of failure on fertility of some semen batches.

Há no organismo humano Glutationas peroxidases (GPx), muitas dessas responsáveis por proteger as células contra espécies reativas de oxigênio (ROS, do inglês Reactive Oxygen Species), cuja detecção pode ser realizada por sondas fluorescentes. Tais sondas podem conter em sua estrutura átomos de selênio ou telúrio. Esses calcogênios apresentam várias propriedades, dentre elas a suscetibilidade a processos oxidativos, que podem levar a mecanismos de transferência eletrônica fotoinduzida (PET), promovendo um sistema on/off de fluorescência na molécula. Sendo assim os derivados de selenoaminoácidos e sondas fluorescentes contendo calcogênios podem ser empregados tanto na eliminação de espécies reativas de oxigênio como na sua detecção. O presente trabalho propôs a síntese de derivados de selenoaminoácidos, via reações do tipo Morita-Baylis-Hillman, que poderiam ser empregados como antioxidante. Entretanto, foi também foco da nossa pesquisa explorar a síntese de novos composto organocalcogênios com propriedades fluorescentes e seu estudo fotofísico / Glutathione peroxidases (GPx) are enzymes present in the human body, and many of them are responsible for protecting cells against reactive oxygen species (ROS), which can be detected by fluorescent probes. Selenium or tellurium atoms can be present in these probes and are susceptible to oxidative processes, which can lead to photoinduced electron transfer mechanism (PET). These mechanisms can promote an on/off fluorescence system, thus, selenium amino acids derivatives and fluorescent probes containing chalcogens can be used both in the elimination of reactive oxygen species and their detection. Herein, we proposed the synthesis of selenium amino acids derivatives, vía Morita-Baylis-Hillman reaction, and the products were pointed out as potential antioxidants. In addition, it was also the focus of our research exploring the synthesis of new organochalcogen compounds with fluorescent properties and their photophysical study.